海鞘细胞核骨架与细胞质骨架连接复合体相关基因的克隆及表达分析

Cloning and Expression Patterns of LINC Complex Component Encoded Genes in Ascidian Ciona robusta

为了研究LINC(Linker of nucleoskeleton and cytoskeleton)复合体相关基因KASH(Klarsicht/Anc/Syne-1)和SUN(Sad1/UNC84)在海鞘(Ciona robusta)细胞核位置决定中的角色和作用,本研究鉴定并分别克隆了海鞘LINC复合体中编码2种Cr_KASH蛋白和2种Cr_SUN蛋白的基因。荧光定量PCR方法分析了上述4个基因在海鞘胚胎各个时期的表达量,发现在胚胎发育的尾芽延伸期,几种基因的表达量均有不同程度的提高。原位杂交方法揭示Cr_KASH1、Cr_SUN1和Cr_SUN2特异表达在海鞘胚胎的脊索组织中,而Cr_KASH2则特异表达在肌肉组织。在NIH/3T3细胞中的亚细胞定位分析证实,上述LINC复合体蛋白与不同的细胞骨架交联。通过破坏细胞骨架微丝和微管蛋白的聚合发现会影响KASH和SUN在细胞中的定位和结构,暗示海鞘LINC复合体成分和细胞质骨架蛋白协同作用调控细胞核的形态和位置。本文研究结果表明,在海鞘不同胚胎发育阶段和不同组织中,细胞核骨架或许通过不同LINC蛋白组分与细胞骨架结合影响细胞核的行为。

In this study,we identified and cloned genes encoded two KASH proteins and two SUN proteins in Ciona genome.Quantitative real-time PCR results showed that expression levels of these genes were increased to different degrees during the embryo development.In situ hybridization revealed that Cr_KASH1,Cr_SUN1,and Cr_SUN2 were specifically expressed in the notochord tissue,while Cr_KASH2 was specifically expressed in muscle tissue in Ciona embryos,suggesting that these proteins were involved in the regulation of nuclear location in different tissues and developmental stages.Subcellular localization of these proteins in the NIH/3T3 cell line showed that these LINC complex component proteins cross-linked to the cytoplasmic cytoskeleton components.Disruption of the polymerization of actin or microtubule filaments affected the localization and structure of Cr_KASH and Cr_SUN in the cells,suggesting that the components of the LINC complex and cytoplasmic skeletal proteins are cooperated in the regulation of the morphology and location of the nucleus.The results of this study indicate that the nuclear skeleton in different Ciona tissues binds to the cytoskeleton through different linker proteins,and they interact and synergistically affect the behavior of the nucleus.